DEPARTMENT OF BIOTECHNOLOGY

M.TECH
IMMUNOTECHNOLOGY LABORATORY
(481172L2)

HOD
 IMMUNOTECHNOLOGY LAB MANUAL

EXPT.NO NAME OF THE EXPERIMENT PAGE NO. SIGN

1. IDENTIFICATION OF CELLS IN BLOOD SMEAR 4

2. IDENTIFICATION OF BLOOD GROUPS 5

3. IMMUNOELECTROPHORESIS 7

4. PURIFICATION OF CHICKEN IGY 9

5. SINGLE RADIAAL IMMUNO DIFFUSION 10

6. DOUBLE DIFFUSION (OUTCHERLONY METHOD) 11

7. HAEMAGGLUTINATION 12

TESTING FOR TYPHOID ANTIGENS BY WIDAL

8. 13
TEST

SEPARATION OF T AND B LYMPHOCYTES BY T-

9. 14
ROSETTE ASSAY

ENZYME LINKED IMMUNOSORBENT ASSAY

10. 15
ELISA

11. IMMUNO ELECTROPHORESIS 18

PREPARED BY
Dr.R.BALACHANDAR

1
IMMUNOTECHNOLOGY LAB MANUAL
 GENERAL LABORATORY PROCEDURES, EQUIPMENT USE, AND SAFETY
CONSIDERATIONS
CHEMICALS
A number of chemicals used in any molecular biology laboratory are hazardous. All
manufacturers of hazardous materials are required by law to supply the user with pertinent
information on any hazards associated with their chemicals. The following chemicals are
particularly noteworthy:
 Phenol - can cause severe burns
 Acrylamide - potential neurotoxin
 Ethidium bromide - carcinogen
 Formaldehyde, Acetonitrile, Chloroform are potentially harmful
These chemicals are not harmful if used properly: always wear gloves when using
potentially hazardous chemicals and never mouth-pipette them. If you accidentally splash
any of these chemicals on your skin, immediately rinse the area thoroughly with water and
inform the instructor. Discard the waste in appropriate containers.

ULTRAVIOLET Light
Exposure to ultraviolet light can cause acute eye irritation. Since the retina cannot
detect UV light, you can have serious eye damage and not realize it until 30 min to 24 hours
after exposure. Therefore, always wear appropriate eye protection when using UV lamps.

ELECTRICITY
The voltages used for electrophoresis are sufficient to cause electrocution. Cover the
buffer reservoirs during electrophoresis. Always turn off the power supply and unplug the
leads before removing a gel.

GLASSWARE AND PLASTIC WARE .

Glass and plastic ware used for molecular biology must be scrupulously clean. Dirty test
tubes, bacterial contamination and traces of detergent can inhibit reactions or degrade
nucleic acid.
Glassware should be rinsed with distilled water and autoclaved or baked at 150 degrees C
for 1 hour. Make sure that the tubes you are using are resistant to the chemicals used in
your experiment. Micro pipet tips and microfuge tubes should be autoclaved before use.

GENERAL HOUSEKEEPING
All common areas should be kept free of clutter and all dirty dishes, electrophoresis
equipment, etc should be dealt with appropriately. Since you have only a limited amount of
space to call your own, it is to your advantage to keep your own area clean. Since you will
use common facilities, all solutions and everything stored in an incubator, refrigerator, etc.
must be labeled. In order to limit confusion, each person should use his initials or other
unique designation for labeling plates, etc. Unlabeled material found in the refrigerators,
incubators, or freezers may be destroyed. Always mark the backs of the plates with your
initials, the date, and relevant experimental data, e.g. strain numbers.

2
IMMUNOTECHNOLOGY LAB MANUAL
DECONTAMINATION of Ethidium Bromide Spills

EtBr spills can be decontaminated with a solution of 20 ml of hypophosphorus acid

(50%) added to a solution of 4.2 g of sodium nitrate in 300 ml water. Prepare fresh solution
the day of use in a fume hood. Wear rubber gloves, lab coat, and safety glasses. Turn off
electrical equipment before decontamination.

 Soak paper towel in decontamination solution, place on contaminated surface, and

scrub.
 Scrub five more times with paper towels soaked in water, using fresh towel each
time.
 Place all towels in a container and soak in fresh decontamination solution for one
hour.
 Test squeezings from final towel scrub and mixture for fluorescence; repeat
procedure
with fresh decontamination solution if fluorescence is present.

 Neutralize with sodium bicarbonate and discard as nonhazardous aqueous waste.

 This procedure has been validated for EtBr-contaminated stainless steel, Formica,
glass, vinyl floor tile surfaces, and filters of transilluminators.
Alcohol solutions that characterize as hazardous wastes are prohibited from discharge to
the sewer.

The waste alcohol must be diluted with plenty of water inorder to reduce the concentration
of the alcohol concentration before its discharge into the sewer.

DISPOSAL OF AGAR/LB PLATES

Any uncontaminated, solidified agar or agarose should be discarded in the trash, not
in the sink, and the bottles rinsed well.Any media that becomes contaminated should be
promptly autoclaved before discarding it. Petri dishes and other biological waste should be
discarded in Biohazard containers which will be autoclaved prior to disposal.

CLEANLINESS OF THE LABORATORY

The gel apparatus should be washed immediately after use. The glass plates must be
rinsed with plenty of tap water, ethanol and finally with distilled water, wipe it with tissue
paper. Beakers, measuring cylinders and all other glass wares should be rinsed with distilled
water before and after use. The working area must be kept clean without any spillage,
broken glass wares before and after the use.

3
IMMUNOTECHNOLOGY LAB MANUAL
 IDENTIFICATION OF CELLS IN BLOOD SMEAR

EXPT NO. 1
DATE :
AIM
To identify different WBC cells present in the blood smear

MATERIALS REQUIRED
Blood sample, microscopic slides, leishmann’s stain, light microscope and double
distilled water.

PROCEDURE
 A drop of blood is placed over the clean glass slide at the centre and spread the
blood to form a smear.
 Air dry the blood smear and label properly
 Apply leischmann’s over the smear and allow for couple of minutes, then add equal
volumes of double distilled water and mix properly.
 Keep the slide aside for 10-20 minutes and wash the slide in running water and air
dry the smear.
 After air drying, observe the blood smear under light microscope.

RESULT
The following cells were identified in the blood smear using light microscope

4
IMMUNOTECHNOLOGY LAB MANUAL
 IDENTIFICATION OF BLOOD GROUPS
EXPT NO. 2
DATE :
AIM

To demonstrate antigen-antibody reaction by Hemeagglutination reaction.

PRINCIPLE

Blood group classification have extreme importance in medical field. ABO blood
groups were discovered by Carl Landsteiner. The system classifies human blood into four
main groups based on the presence or absence of the cell surface antigens.

 If the red cells have only antigen A at its surface, the blood type is ‘A’ where the
plasma contains Anti-B antibodies which clumps the cells.
 If the red cells have only Antigen B, the blood is type ‘B’ where the plasma contains
Anti-A antibodies, which clumps cells having antigen A.
 If the cells have both antigen A and antigen B, the plasma contains neither Anti-A or
Anti-B.
 Rh blood types form the second major blood group system. Rh factor agglutinate
with Anti-Rh antibody. This reaction can produce serious illness or death. When the
blood agglutinates with Anti-Rh antibody, it indicates the person is Rh positive and if
there is no agglutination, it indicates the person is Rh negative.
ABO blood grouping has tremendous application in the field of blood transfusion, tissue
typing, graft transfer and other medical ailments.

MATERIALS REQUIRED

Blood sample, antiserum (anti-A, anti-B, anti-D), glass slide, lancet or needle,
rectified spirit and cotton.

PROCEDURE

 Label three glass slides as A,B, and D .

 Add one drop of blood to each labeled areas.
 Add a drop of antisera-A to labeled A, a drop of antisera-B to labeled B and a drop of
antisera D to labeled D.
 Mix the antisera with blood slowly and properly using the edges
 After mixing, observe for agglutination if any.
OBSERVATION

 Aggregates of blood sample is observed in the labeled glass slide.

INTERPRETATION

 Agglutination of blood with antisera occurs. If agglutination occurs at glass slide A, it

is A blood group.

5
IMMUNOTECHNOLOGY LAB MANUAL
  If agglutination occurs at glass slide B, it is B blood group.
 If there is no agglutination in glass slide A and B, it indicates O blood group.
 Agglutination at glass slide D indicates the presence of Rh factor.
RESULT

The blood sample was analyzed and the blood group is found to be______

6
IMMUNOTECHNOLOGY LAB MANUAL
 IMMUNO ELECTROPHORESIS
EXPT NO. 3
DATE :
PRINCIPLE:

Immunoelectrophoresis consists of two methods that are combined, namely agar-gel

electrophoresis and immunodiffusion. The agar plate has a longitudinal trench cut which
accommodates the antiserum after electrophoresis and a central well or wells to
accommodate test material. The serum sample is placed in the well or wells and
electrophoresis is carried out for 1-2h at a field strength of 5-9 V/cm. The protein
components separate according to their charge, molecular weight and shape but unlike
normal electrophoresis, the bands are not stained for proteins. Instead antiserum is
pipetted into the longitudinal trough and the plate is incubated in humid chamber for
overnight. Diffusion of the antiserum and protein occurs and, where they meet, precipitin
arcs are formed that give rise to a characteristic pattern.

MATERIALS:

1. 0.07M Baribiturate buffer; pH 8.6 (Dissolve 2.58g of diethylbarbituric acid and 14.42g
of sodium diethylbarbiturate in water and make up to 1litre).
2. Agarose (medium EEO) solution (1% w/v in the barbiturate buffer: heat the agarose
at 900C until all the lumps are dissolved and clear solution is obtained and pour on to
a slide when it is cooled to say 55 0C. Allow the agarose to solidify.)
3. Glass slides (7.5 cm x 5cm).
4. Incubator at 370C
5. Horizontal electrophoresis apparatus
6. Power supply
7. Cords
8. Gel puncher
9. Shandon cutting device
10. Bromophenol blue
11. Saline (0.9%w/v NaCl; 0.1w/v Na azide)
12. Antigen
13. Rabbit antiserum or peak fraction of the first peak in the previous experiment.
14. 0.25% coomassiee brilliant blueR 250 prepared in 4:1:5 (v/v/v) in ethanol: acetic acid:
distilled water.
15. Destain solution (4:1:5 (v/v/v) in ethanol: acetic acid: distilled water).
METHOD:

 Heat glass plates in an oven at 100 0C for about 30 minutes then remove them and
place on a level surface.
 Carefully pipette about 8 ml of the hot agarose solution on to the surface of the
slides.
 The molten agarose will set in a uniform layer and the surface tension will hold the
agarose at the edges. Allow the agarose to solidify.

7
IMMUNOTECHNOLOGY LAB MANUAL
  Punch the gel with one well of relatively bigger size and with the help of Shandon
cutting device give 4.5 cm long trough of 1mm diameter. With the help a of sharp
needle remove the agarose from the well as well as from the trough.
 The antigen is mixed with 0.025% bromophenol blue, the tracking dye. Fill the
electrophoresis chamber with 0.07M barbiturate buffer and place the slide in the
chamber and connect the slide with wigs prepared from Whatman No 3 filter papers
on either side and the wigs must dip into the buffer solution.
 Apply the potential difference of 5-9 V/cm for about 1-2h or until the tracking dye
reaches the 2.0 -2.5 cm from the well.
 Remove the slide from the electrophoresis chamber and add of the antiserum or
purified IgG place the slide in the moist chamber and incubate for overnight at 37 0C
and observe the precipitin line formation on the following day.
OBSERVATION

 The appearance of the precipitin line is noticed in the presence of specific antibody.
INTERPRETATION

 Immunoelectrophoresis is a diagnostic tool for qualitative and quantitative

determination of biological samples.
 The specificity of the antigen can be ascertained to the specific antibody based on
the pattern of precipitin arc.

RESULT

Thus the specific of the antigen towards the specific antibody is ascertained.

8
IMMUNOTECHNOLOGY LAB MANUAL
 PURIFICATION OF IMMUNOGLOBULIN (IGG FROM EGG YOLK)

EXPT NO. 4
DATE :
AIM

To purify chicken immunoglobulin G from egg yolk.

Principle

Chicken IgG is also known as IgY. It is the major serum antibody. It is transported to
the egg similar to placental transfer of IgG in mammals. This experiment works on the
mechanism of precipitation of organic polymers. Organic polymers dicrease the dielectric
constant of solution and hence its solvating power. Thus the solubility of protein can occur
due to electrostatic attraction. Precipitation occurs when pH is close to isoelectric point of
the protein.

REQUIREMENTS

Chicken egg, glasswares, magnetic stirrer/ magnetic pellet, sterile syringe, gauze
cloth, precipitation solution, PBS, SDS-PAGE reagents and solutions.

PROCEDURE

 The egg yolk is initially warmed to room temperature.

 The egg is carefully cracked under sterile conditions and its contents are poured in a
sterile petri dish, without disturbing the yolk.
 The egg white was separated completely using syringe (without needle)
 The egg yolk was poured in beaker and its weight was measured.
 The yolk was stirred at room temperature with slow addition of 3 volumes of
precipitation solution.
 Strring was continued to precipitate the lipids.
 This solution was centrifuged at 10000rpm for 15 minutes.
 The supernatant was collected by filtering through fine gauze cloth.
 The volume of supernatant was measured and 1/3 volume of ammonium sulphate
was added to it.
 The mixture was stirred for 10 minutes to precipitate IgG.
 The solution was centrifuged at 10000rpm for 15min at 4C.
 Supernatant was discarded and pellet was washed with. PBS and finally suspended in
PBS.
 SDS-PAGE analysis of purified IgG was carried out.
RESULT

The immunoglobulin-Y (IgY) was purified from the chicken egg yolk and fractionated
using SDS-PAGE.

9
IMMUNOTECHNOLOGY LAB MANUAL
 SINGLE RADIAL IMMUNO DIFFUSION (SRID)
EXPT NO. 5
DATE :
AIM

To quantify the amount of antigen present in test sample by single radial immuno diffusion.

PRINCIPLE

Radial immuno-diffusion is a method for determining the concentration of an

antigen. The antigen sample is placed in a well and allowed to diffuse into agar containing a
suitable dilution of an anti-serum. The area of the precipitin ring that forms around the well
in the region of equivalence is proportional to the concentration of the antigen.
The antigen-antibody precipitation is made more sensitive than in double immuno
diffusion by the incorporation of the antiserum in the agarose solution before the gel is
made. Thus the antibody remains uniformly distributed throughout the gel. Antigen is then
allowed to diffuse from wells cut in the gel. This is an example of single immuno diffusion.
Initially as the antigen diffuses out of the well, the antigen concentration is relatively
high and soluble antigen-antibody adducts are formed. However, as antigen diffuses farther
from the well, the antigen-antibody complex reacts with more amount of antibody resulting
in a lattice that precipitates to form a precipitin ring. The concentration of antigen is
directly proportional to the diameter of the precipitin ring.
REQUIREMENTS

Antigen, antibody, test antigen. Agarose , gel punch, class slide, saline, template.
Titer plates, micropipette, glasswares etc.,

PROCEDURE

 1% agarose was prepared in saline and heated to dissolve the agarose.

 At optimum temperature (~40C), antibody of desired volume is added to agarose 3-
4mm of agarose gel is prepared on microscopic slide.
 The slides were stored at 4C for a short time.
 After agarose has hardened sufficiently, two sets of wells, 3 on top and3 on bottom
was cut using gel punch with the help of template.
 The antigen was diluted two fold in micro titer plate.
 Antigen is serially diluted in normal saline to obtain 1:2, 1:4,1:8,1:16 dilutions.
 10ul of undiluted antigen was added to the first well of the agarose gel.
 10ul of serially diluted antigen was added to 2 nd, 3rd, 4th and 5th wells.
 10ul of sample antigen was added to the 6 th well.
 After filling the wells, the slide was kept in a flat bottom container whose interior
was kept moist by using damp cotton.
 The slides were placed at room temperature for 18-24 hours.
 Diameter of precipitin ring was measured and calibration curve was plotted.
RESULT

The antibody titration at various dilution was carried out and the concentration of
the test antigen was found to be

10
IMMUNOTECHNOLOGY LAB MANUAL
 DOUBLE DIFFUSION (OUTCHERLONY METHOD)
EXPT NO. 6
DATE :
AIM

To quantitate and characterize antigen and antibodies by ouchterlony double

diffusion

PRINCIPLE

Immunodiffusion in gel encompasses a variety of techniques, which are useful for

the analysis of antigen and antibodies. One such method is ouchterlony double diffusion.
Both qualitative and quantitative information can be obtained from double diffusion assays.
This techniques is used for comparing different Ag preparations.
In the ouchterlony method, both antigen and antibody diffuse radially from wells
towards each other, thereby establishing a concentration gradient. As equivalence is
reached, a visible live of precipitation is formed. This simple technique is an effective
quatitative tool for determining the relationship between antigens and the number of
different antigen-antibody systems present. The pattern of the precipitin lines that form,
where two different antigen preparations are placed in adjacent wells indicates whether
they share epitopes. The pattern of lines that form, can be interpreted to determine
whether, the antigens are same or different.
REQUIREMENTS

Microscopic slide, template, gel punch, saline, agarose, antigens and antibodies, test
serum, glasswares etc.,

PROCEDURE

 1% agarose was prepared in normal saline and heated to polymerize the agarose
 3-4mm thick agarose gel was prepared on glass slide
 The slides were stored at 4C for a short time.
 After agarose has hardened sufficiently, two sets of wells were cut using gel punch
with the help of template.
 Antibody and the test serum was diluted to two fold and used.
 In the first set well, antigen was added in central well and diluted antibody was
added in surrounding wells (1:2 to1:16 dilutions)
 In second set, antigen was added to central well and diluted test serum was added in
surrounding wells.
 After filling the wells, the slide was kept in a flat bottom container whose interior
was kept moist by using damp cotton.
 The slides were placed at room temperature for 18-24 hours.
RESULT

The antibody titration at various dilution was carried out and the variation in the
precipitin arc was observed.

11
IMMUNOTECHNOLOGY LAB MANUAL
 HAEMEAGGLUTINATION
EXPT. NO. 7

DATE .

AIM

To perform agglutination reaction

PRINCIPLE

Haemagglutination is a convenient and simple technique. Some viruses have the

property of adsorbing to red blood cells through the receptor sites on their surface resulting
to form haemagglutination, i.e., clumping of red blood cells.

MATERIALS REQUIRED

1%Chicken RBC, micropipettes, glass wares, distilled water.

PROCEDURE

Preparation of virus (HA antigen)

 Dissolve the virus in freeze dried vial with 1ml of diluent and mix it properly. Store at
4°C.
Preparation of 1% chicken erythrocytes (CRBC)

 Collect chicken blood from healthy chicken in Alsever’s solution and carefully
centrifuge at 2000 rpm for 5 minutes to pack the red blood cells.
 Discard the supernatant. Wash the CRBC by adding 10ml of saline and mix it well.
 Again centrifuge at 2000 rpm for 5 minutes and discard the supernatant, repeat the
procedure for three times.
 After discarding the supernatant, the sedimented RBC’s are reconstituted to a final
concentration of 1% in normal saline.
 Add 25μl of normal saline to all the wells in ‘A’ arrow of a V bottom 96 well
microtitre plate.
 Add 25μl of antigen to the first well and carry out serial dilution.
 The last well acts as control (RBC control)
 Add 25μl pf normal saline to all the wells.
 Add 50μl of 1% chicken RBC to all wells of the microtitre plate.
 Gently tap the microtitre plate for efficient mixing.
 Wrap it with aluminium foil and keep the microtitre plate at 4 °C for the settling of
RBC’s .
RESULT

The titre value of the virus can be ascertained and the titre value is found to
be___________

12
IMMUNOTECHNOLOGY LAB MANUAL
 TESTING FOR TYPHOID ANTIGENS BY WIDAL TEST
EXPT.NO. 8

DATE.

AIM

To diagnose the suspected case of Enteric fever serologically

PRINCIPLE

Widal test is serological method used in the diagnosis of Enteric fever which is
caused by the organisms of the genus salmonella. They are salmonella typhi, Salmonella
Paratyphi A,B,C,D, etc.

Enteric fever due to S.typhi and S.Paratyphi A,B,C and D are common in India.
Antigens specifically prepared fromthis orgaism are used in the agglutination test to detect
the presence of antibodies in patient sera which are elucidated in response to infection by
these bacteria.

The organisms causing enteric fever possess two major antigens namely somatic ‘O’
antigen and flagellar “H’ antigen along with another ‘VI” surface antigen. During infection,
antibodies are produced in patients sera against these ‘O’, ‘H’ S.typhi , ‘A(H)’ and ‘B(H)’
S.paratyphi antigens.

MATERIALS REQUIRED

Six circle glass slides, disposable mixing sticks, positive patient serum, micropipette.

PROCEDURE (Rapid Slide Test-Screening test)

 Take a properly cleaned glass slide

 Add a drop of undiluted test serum in the corresponding reaction circles and a drop
of positive and negative control serum in the circles marked positive control and
negative control.
 Add a drop of antigen O,H, A(H) amd B(H) in respective circles.
 Add any one of ht ‘H’ antigen [H,A(H), or B(H)] in the positive and negative control.
 Mix the contents of each circle with separate disposable mixing sticks.
 Keep the slide for one minute and then observe the visible agglutinins.

RESULT

13
IMMUNOTECHNOLOGY LAB MANUAL
 SEPARATION OF T AND B LYMPHOCYTES FROM HUMAN PERIPHERAL
BLOOD(T-Cell Rosseting method)
EXPT.NO.9

DATE.

AIM:

To separate T & B lymphocytes from peripheral blood by E-rosette technique.

PRINCIPLE:

Human T cells have specific surface antigen CD-2 on this surface. This has a natural
affinity for a sheep erythrocyte on incubating isolated lymphocytes with sheep blood. If will
get attached to the receptor on T cell and under microscope it will look like floral structure
called rosette to three or mare sheep RBC.

MATERIALS REQUIRED:

 Isolated sample lymphocytes

 Sheep blood collected in alsever’s solution
 Centrifuged tubes
 Haemocytometers.
PROCEDURE:

1. Human and sheep blood was taken in 1:1 ratio and incubated for 10 minutes at 37°
C.
2. A drop of suspension was taken under microscope for rosette.
3. Centrifuge the suspension at 1000 rpm for 10 minutes to sediment the rosette.
4. Collect the supernatant containing B-cells and counted in haemocytometer.
5. Lyse the sheep RBC’s and count the ‘T’ cells in haemocytometer.
OBSERVATION:

Flower like rosette were seen on. Incubation of isolated lymphocytes with sheep
RBC.

RESULT:

Human T-cell and B-cell lymphocytes were separated by E-rosette technique.

14
IMMUNOTECHNOLOGY LAB MANUAL
 ELISA (SANDWICH)
EXPT.NO. 10

DATE.

AIM

To perform quantification of antigen using ELISA

MATERIALS REQUIRED

Titer plate, micropipette, glasswares, PBS, saline, primary antibody, antigen,

conjugate , substrate, blocking buffer, washing buffer, substrate buffer, coating buffer,
distilled water.

1. Coating Buffer

Solution A - 1.0 M NaHCO3 8.4 g / 100ml

Solution B - 1.0 M Na2CO 10.6 g / 100ml.
Mix 45.3 ml Sol A
18.2 ml Sol B
Adjust the pH to 9.6
2. 10 X Phosphate Buffered Saline (PBS).

KCl 2.0 g
NaCl 80.0 g
Na2HPO4. 7 H2O 21.7 g
KH2PO4 20.0 g
Add distilled water upto 1 litre and adjust the pH to 7.4 with 10 N NaOH.

3. Blocking solution

10 X PBS pH 7.4 50 ml
0.05% Tween -20 0.25 ml
5% BSA 25 g.
Add distilled water up to 500 ml, filter

4. ELISA Diluent

10 X PBS pH 7.4 100.0 ml

0.05% Tween - 20 0.5 ml
1% BSA 10.0 g.
Add distilled water up to 1 liter, filter.

5. Washing Buffer

10 X PBS pH 7.4 400 ml

0.05% Tween-20 2 ml.
Add distilled water and make up to 4 liters.

15
IMMUNOTECHNOLOGY LAB MANUAL
6. Substrate Diluent

NaHCO3 1.69 g
Na2CO3 2.51 g
Mg Cl2 0.41 g.

Add distilled water and make up to 2 liter, pH 8.6.

PROCEDURE

ANTIBODY COATING


The antibody was diluted in coating buffer provided at 1:100 dilutions and 100ul of
antibody was added to each well of ELISA plate
 The plate was left at 4C overnight for passive absorption of antibody to the ELISA
plate
WASHING

Unabsorbed antibody need to be removed after overnight incubation

Washing is done using ELISA washer or manually by adding 300ul of washing buffer
to each well and discarding it.
 Manual washing is repeated thrice after which the plate is flipped to completely
drain the washing buffer.
BLOCKING

 Blocking buffer was then added to the wells (300µl/well). Blocking buffer was added
to prevent cross reactivity.
 Proteins in blocking buffer (BSA) bind to unbound sites in microtiter plate, so that
these sites are not exposed during the course of the reaction process.
 The plate was incubated at 37°C for 1 hours.
 After 1 hour manual washing was repeated thrice.

ANTIGEN DILUTION


The antigens that need to be quantified is serially diluted in PBS.

Serially double dilution from 1:100 to 1:1600 was performed.

Similarly the sample antigen was diluted in 1:100 in PBS.

Diluted standard and sample antigen was added in duplicated. A set of well was left
empty which is the blank.
 The plates were incubated at 37C for 1 hour.
CONJUGATE ADDITION

 After incubation, the plate is washed thrice manually and 100ul of diluted conjugate
(detection antibody/ enzyme tagged) was added to all wells and incubated at 37C for
1 hour.
 Washing step was repeated following incubation

16
IMMUNOTECHNOLOGY LAB MANUAL
SUBSTRATE ADDITION

 100ul of diluted substrate was added to all wells of ELISA plate and the plate was
incubated at 37°C dark for 10 min’s.
 The reaction was then stopped by addition of 25ul of stopping solution.

RESULT:

17
IMMUNOTECHNOLOGY LAB MANUAL
 IMMUNOFLUORESCENCE PROCEDURE

EXPT.NO. 11

DATE.

PRINCIPLE

Immunofluorescence is a technique used for light microscopy with a fluorescence

microscope and is used primarily on microbiological samples. This technique uses the
specificity of antibodies to their antigen to target fluorescent dyes to
specific biomolecule targets within a cell, and therefore allows visualization of the
distribution of the target molecule through the sample. The specific region an antibody
recognizes on an antigen is called an epitope. There have been efforts in epitope mapping
since many antibodies can bind the same epitope and levels of binding between antibodies
that recognize the same epitope can vary. Additionally, the binding of the fluorophore to
the antibody itself cannot interfere with the immunological specificity of the antibody or the
binding capacity of its antigen. Immunofluorescence is a widely used example
of immunostaining (using antibodies to stain proteins) and is a specific example
of immunohistochemistry (the use of the antibody-antigen relationship in tissues). This
technique primarily makes use of fluorophores to visualise the location of the antibodies.

Samples:
1. Cultured cells: Cells may be grown on a 12mm round coverslips and stained in the
wells of a 24-well plate. Alternatively cells can be grown in a petri dish, in which a
hole has been made and a coverslip is glued in (Mat-Tek Corp. 200 Homer Ave,
Ashland MA 01721, (800) 834-9018). Finally, now that we have an upright confocal
microscope cells maybe grown on plastic and viewed live with the Zeiss LSM 510
Meta using dipping objecives.
2. Sections of fixed tissues: Sections up to 200 µm thick made with a Vibrotome can be
viewed on a 2-photon microscope. For standard confocal (single photon), 50-100 µm
should be max.
3. Thin sections: These should be made as usual for histological staining. After
attaching to glass slide (coated with poly-L-lysine or purchased charged slides: Vector
Labs), they must be fixed.

Fixation:

Since different fixatives can have various effects on cytoskeletal structures or on proteins by
cross-linking, several methods of fixation should be tested for each antibody developed:

1. 3.7% paraformaldehyde (PFA) in PBS for 15 minutes at room temperature followed

by 0.2% Triton X100 in PBS (5 min) or 100 µM digitonin (5-10 min) permeabilization.
This is the fix of choice if it works with your antibodies, because the cytoskeleton is
best preserved. (Permeabilization may not be necessary in very thin sections.)
2. Absolute methanol at -20°C for 6 minutes; flood with PBS-0.5% BSA to prevent
drying when incubation is finished.

18
IMMUNOTECHNOLOGY LAB MANUAL
 3. Absolute ethanol at room temperature for 30 seconds; flood with PBS-0.5% BSA to
prevent drying when incubation is finished.

Fluorescence staining:
1. Wash the cells several times in PBS-0.5% BSA. (I just use a squirt bottle and
thoroughly flush the slide several times or fill and dump in the case of cells grown on
coverslips and incubated in 24-well plates.)
2. Incubate with primary antibodies in PBS-0.5% BSA for 1 hour at room temperature or
a half hour at 37°C. I usually only make enough working dilution for each experiment
and don't reuse it or save left over. The total amount I use for 24-well plates or for
circles drawn on slides is 10µl. But I don't measure less than a microliter of stock; so
if it should be diluted 1:200, I make 200 µl. If there are two antibodies I mix them
first, then place the solution on the cells. Whether on a slide or on coverslips in wells
this incubation should be done in a humid chamber. This can be accomplished by
placing the slide on toothpicks on a damp paper towel in a plastic box of some sort,
or placing the damp paper towel in the lid of the 24-well plate.
3. After incubation, wash again in PBS, and add fluorescent secondary antibodies (at
this point I add the direct labeling reagents, eg DAPI or phalloidin, as well), followed
again by washing. Only secondary antibody should be incubated on one sample to
control for nonspecific reaction. Incubate and wash again as above.

Mounting:

They are embedded in an anti-fade media (available from Molecular Probes or

Vector labs). There are of course many variations on this media. It is only important
that the issue of fading be addressed somehow. I will often rinse my cells for
preparation once in deionized water before mounting too. This simply rinses off any
salt residue, but is not necessary for viewing. Thick sections of 60-200 µm may be
stored in a PBS a solution containing 2% DABCO instead of mounting.

RESULT:

19
IMMUNOTECHNOLOGY LAB MANUAL